Uncovering microbial hydrocarbon degradation processes: the promise of stable isotope probing

Traditional microbiological methods for the identification of microorganisms after they have been isolated in pure culture have revealed key players in the degradation of hydrocarbons. But have we identified them all? The conspicuous enrichment of an uncultured Oceanospirillales in a sub-surface hydrocarbon plume during the Deepwater Horizon oil spill is one of many examples highlighting that we are not there yet in this respect. Culture-dependent methods typically miss identifying 99% of microorganisms originating from environmental samples, and are on their own ineffective in resolving the diversity and function of natural microbial communities. Stable isotope probing (SIP) is a technique used to identify a target group of microorganisms which can actively metabolize a specific substrate in an environmental sample and, thus, under in situ-like conditions. The technique involves incubating an environmental sample with an isotopically-labeled (e.g., ¹³C, or ¹⁵N) substrate and allowing the label to become incorporated into the biomass (e.g. DNA, RNA, protein, PLFAs) of those microorganisms capable of metabolizing the substrate. The labeled biomolecules are then isolated and analyzed to identify the organisms that actively incorporated the isotope label. SIP based on DNA or RNA are quite similar methods by the nature of their execution, albeit with subtle differences. The technique has a high phylogenetic resolution, and has provided many new insights to this day concerning microbial biodegradation of specific compounds and putative interrelationships of microbial activities with biogeochemical processes. This chapter provides an overview on the methodology, its caveats, and gives examples of applications for exploring the diversity of microbial hydrocarbon degraders in seep and other benthic habitats.